Project Summary This research proposal focuses on structural properties that govern the functional behavior of the nitric oxide synthase (NOS) isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), in their respective environments. Three genes encode NOS enzymes, and other products of these genes are expressed in various tissues as a result of alternative RNA splicing. L-Arginine is the natural substrate for NOS isoforms, producing L-citrulline and NO, which serves as a gaseous messenger in the processes of neurotransmission, cytotoxicity or vasodilatation, among others, depending upon the isoform and tissue localization. The hypotheses to be addressed in this proposal are that the molecular design of the NOS isoforms, while requiring the same cofactors and prosthetic groups (FAD, FMN, Fe-protoporphyrin IX, Zn and tetrahydrobiopterin), is adapted in each isoform to satisfy its specific cellular function. For example, sequence inserts in the constitutive NOS enzymes (nNOS and eNOS) confer regulatory properties that do not exist in the inducible isoform (iNOS). Therefore, molecular studies will be focused on further examination of structural properties, using new techniques to examine intrinsic regulatory elements and relationships and the determination of mechanisms that bear on O2 metabolism. Specific Aim 1: To determine the structural properties of the NOS holoenzymes and derivative domains, using crystallography and cryo-electron microscopy; Specific Aim 2: To address intrinsic regulation of the nNOS and eNOS by nuclear magnetic resonance spectroscopy, laser flash photolysis and protein film voltammetry to determine the mechanistic properties of these proteins; and Specific Aim 3: To address the mechanisms involved in O2 metabolism and the process of oxygenation, using rapid-freeze-quench ENDOR, and conventional O2 metabolism measurements. These experiments will test the following hypotheses: 1) that the overall architecture of each NOS isoform determines the intrinsic control of electron transfer through these enzymes; 2) that these isoform-specific architectural features influence their catalytic regulation; and 3) that differential O2 metabolism is also based on structural features of the three isoforms.